Genetically Engineering Plants for Virus Resistance
Plant viruses are a major problem in agriculture and cause significant losses in crop yield each year. In the past, available approaches for combating plant viruses were primarily limited to the selection of plant lines which exhibited genetic resistance to virus infection and the application of chemicals designed to protect plants from the organisms responsible for introducing the virus to the plant (i.e. viral vectors).
Recently, a number of approaches for combating plant viruses have been developed which are based upon the transformation of susceptible plant species with chimeric genes which express transcripts or proteins that inhibit viral infection. These approaches include genetically engineering plants to express viral coat protein or coat protein transcripts, viral replicases in unmodified or modified form, antisense genes or ribozymes targeting viral genomnic RNA or transcripts, and altered viral transcripts (for a review, see Fitchen, J. H. et al., Ann. Rev. Microbiol. 47. 739-763 (1993)). To apply any of these approaches, knowledge of the structure and organization of the genome of the target virus is necessary.
With respect to the expression of altered viral transcripts to confer viral resistance, limited success has been reported in dicotyledonous plants through the expression of viral coat protein transcripts which have been modified to render them incapable of translation. Expression of such "untranslatable" viral transcripts in tobacco has been reported to inhibit tobacco etch virus (Lindbo, J. A. et al., Mol. Plant-Microbe Int. 5(2): 144-153 (1992); Lindbo, J. A. et al., Virology 189: 725-733 (1992); PCT application publication no. WO 93/17098 to Dougherty, W. G. et al. (Sep. 2, 1993); Lindbo, J. A. et al., The Plant Cell 5: 1749-1759 (1993)), tomato spotted wilt virus (Pang, S. et al., Biotechnology 11: 819-824 (1993); DeHaan et ai, Bio/Technology 10: 1133-1137 (1992) and potato virus Y (Van der Vlugt, R. A. et al., Plant Mol. Biol. 17: 431-439 (1991).
The ability of such untranslatable RNAs to inhibit viral infection does not appear to be universal, however. Failure of such altered viral transcripts to inhibit viral infection have been reported for tobacco mosaic virus (Powell, P. A. et al., Virology 175: 124-130 (1990) and zucchini yellow mosaic virus (Fang, G. et al., Mol. Plant-Microbe Int. 6(3): 358-367 (1993), a potyvirus similar to tobacco etch virus. Additional unreported failures may also exist, since such negative results are rarely published.
Maize Dwarf Mosaic Virus
Maize dwarf mosaic virus (MDMV) is classified as a member of a group of plant viruses known as the potyviruses. The potyviruses are the largest group of plant viruses and are characterized by a long, flexuous rod particle morphology and are non-persistently transmitted by aphid vectors (see Hollings, M. and Brunt, A., pages 732-807 of "Handbook of Plant Virus Infection and Comparative Diagnosis", ed. by E. Kurstak, pub. by Elsevier/North Holland Biomedical Press, Amsterdam (1981)). The potyviruses have a genome composed of a single strand positive sense messenger RNA molecule which is transcribed and translated as one polyprotein that is subsequently cleaved into its component parts.
MDMV is a major crop pest in maize where it causes mosaic symptoms and dwarfing of infecting plants, ultimately reducing crop yields (Knoke, J. K. et al., pages 235-281 of "Diseases of Cereals & Pulses", volume I, ed. by Singh, U.S. et al., pub. by Prentice Hall, Englewood Cliffs, N.J. (1992)). When found in combination with maize chlorotic mottle virus (MCMV), a synergistic condition known as corn lethal necrosis results causing even more severe crop damage (see Uyemoto, J. K., pages 141-143 of "Proc. Int'l. Maize Virus Disease Colloq. & Workshop", ed. by Gordon, D. T. et al., pub. by Ohio State Univ. and Ohio Agric. Res. Dev. Center, Wooster, Mass. (1983).
The economic impact of yield losses due to MDMV has generated considerable interest in developing strategies to combat this virus. To date, however, only limited success has been achieved in reducing the adverse impact of this virus. Thus there remains a need to identify additional effective means for protecting host plants from MDMV.
The genomic structure and organization of MDMV has remained largely uncharacterized except for the elucidation of viral coat protein coding sequences (see Frenkel, M. J. et al. J. Gen. Virol. 72:237-242, (1991); see also Murray, L. E. et al., Bio/Technology 11: 1559-1564 (1993)). As a result, it is currently not possible to apply many of the more recent recombinant-DNA based approaches that have been used for combating plant viruses to MDMV. These approaches require a more extensive understanding of the structure and organization of the genome of the target virus than is currently available for MDMV.